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☑ Cell treatment (CT)
Western blot analysis of IRG1 on different lysates with Rabbit anti-IRG1 antibody (HA722819) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution.
Lane 1: RAW264.7 cell lysate
Lane 2: RAW264.7 treated with 0.1μg/mL LPS for 6 hours cell lysate
Lane 3: J774A.1 cell lysate
Lane 4: J774A.1 treated with 1μg/mL LPS for 24 hours cell lysate
Lane 5: mBMMC cell lysate
Lane 6: mBMMC treated with 0.1μg/mL LPS and 10ng/mL IFN-gamma for 24 hours cell lysate
Lane 7: mBMSC cell lysate
Lane 8: mBMSC treated with 40ng/mL MCSF for 7 days then add 100ng/mL LPS for 48 hours cell lysate
Lysates/proteins at 10 µg/Lane.
Predicted band size: 54 kDa
Observed band size: 54 kDa
Exposure time: 10 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722819) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
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☑ Cell treatment (CT)
Immunocytochemistry analysis of RAW264.7 cells treated with 0.1μg/mL LPS for 6 hours labeling IRG1 with Rabbit anti-IRG1 antibody (HA722819) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-IRG1 antibody (HA722819) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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